(1) Field of the Invention
This invention generally relates to the field of cancer and, more particularly, to a method and compositions for diagnosing the malignancy of a tumor as well as to compositions and methods for suppressing tumor growth.
(2) Description of Related Art
The E1 a region of human adenovirus types 2 and 5 encodes two major proteins of 289 amino acids and 243 amino acids (289k and 243R) that differ only by an internal 46 amino acid region unique to the 289R protein. These E1A proteins immortalize primary animal cells and cooperate with other cellular and viral oncogenes in oncogenic transformation. These activities are dictated by the binding of several cellular proteins with the E1A proteins. For example, the N-terminal half of E1A proteins, which is encoded by exon 1 of the E1a gene, interacts with cellular growth-regulatory proteins such as the retinoblastoma gene product (pRb) and related proteins (p107 and p130), as well as p300, a CREB binding protein-related transcription factor implicated in transcriptional repression of certain genes (Moran, supra; Eckner et al., Genes Dev. 8:867-884, 1993; Arany et al., Nature 374:81-84, 1995; Lundblad et al., Nature 374:85-88, 1995). The interaction between E1A proteins and the cellular proteins pRb, p107 and p130 cause these cellular proteins to release the E2F transcription factor, thus activating gene expression. For review, see Dyson and Harlow, Cancer Surveys 12: 161-195, 1992; Nevins, J. R., Science 258: 424-429, 1992; Moran, E., Curr. Op. Gen. Dev. 3, 63-70, 1993; Mymryk, J. S., et al., Int. J. Onc. 4, 2131-2141, 1994. Interaction of E1A proteins with p300 releases C-CAF, a cellular acetyl transferase, from the p300/C-CAF complex resulting in activation of transcription by chromatin remodeling (Yang et al., Nature 382:319-324, 1996). Thus, the transforming activities encoded by exon 1 of the adenovirus E1a region appear to be linked to interactions with cellular proteins and the resulting regulation of transcription.
Although the functions of exon 2-encoded domains of E1A proteins have been studied less intensively, these domains have been implicated in certain positive and negative transcriptional regulatory activities (Linder et al., Oncogene 7:439-443, 1992; Bondesson et al., EMBO J. 11:3347-3354, 1992; Mymryk, J. S. et al., J. Virol. 67 6922-2928, 1993). Exon 2 is required for immortalization (Subramanian, T., et al., Oncogene 4:415-520, 1989; Quinlan, M. P. et al., J. Virol. 66:2020-2040, 1992) and induction of Ad2/5-specific cytotoxic lymphocytes (Urbanelli et al., Virol. 173:607-614, 1989). In addition, exon 2 influences the extent of oncogenic transformation. Deletions within the C-terminal 67 amino acids of the E1A 243R protein enhance E1A/T24 ras cooperative transformation (Subramanian, supra; Douglas, J. L., et al., Oncogene 6:2093-2103, 1991), and tumorigenesis of transformed cells in syngeneic and athymic rodent models (Subramanian, supra). Importantly, exon 2 also plays a role in tumor metastasis. Expression of wt E1A efficiently suppresses the metastatic potential of tumor cells (Pozzatti, R. et al., Science 232:223-227, 1986; Pozzatti, R., et al., Mol. Cell. Biol. 8:2984-2988, 1988, Steeg et al., Cancer Res. 48:6550-6554, 1988). In contrast, cells expressing E1A proteins lacking the C-terminal 67 amino acids are highly metastatic (Linder et al., supra; Subramanian, supra). Thus, the E1A protein region encoded by exon 2 appears to negatively modulate in vitro transformation, tumorigenesis and metastasis. These activities have been localized within a 14 amino acid region (residues 225 to 238) near the C-terminus of the 243R protein (Boyd, J. M., et al., EMBO J. 12:469-478, 1993). These transformation restraining activities of the C-terminal region of E1A correlate with the interaction of a 48 kD cellular phosphoprotein termed CtBP (E1A C-terminal Binding Protein) (Boyd et al., supra). CtBP binds to E1A proteins via a 5 amino acid motif, PLDLS, which corresponds to residues 233-237 of the 243R protein. E1A mutants having amino acid substitutions within this motif do not form complexes with CtBP (Schaeper, U., et al., Proc. Natl. Acad. Sci. 92:10667-10671, 1995) and do not have oncogenesis-restraining activities (Schaeper et al., J. Biol. Chem. 273:8549-8552, 1998).
Although interaction of adenovirus E1A proteins with the cellular protein CtBP appears to lead to suppression of tumorigenesis and tumor metastasis of cells transformed with E1A and activated T24 Ras oncogene over expression of CtBP by itself does not appear to exert a significant effect on tumorigenesis and tumor metastasis. One explanation for the inability to detect the effect of CtBP activity could be the fact that expression levels of endogenous CtBP are relatively high. CtBP was found to be abundantly expressed in a variety of human and mouse tissues as well as tissue culture cell lines (Boyd et al., supra). If CtBP functions in a complex with other cellular proteins, overexpression of recombinant CtBP may not cause an effect if cellular cofactors are rate limiting. Thus, to understand the role of CtBP in modulating oncogenesis it would be useful to identify and characterize cellular proteins that interact with CtBP.
Briefly, therefore, the present invention is directed to the identification and isolation of substantially purified proteins that bind to the cellular protein CtBP. Accordingly, the inventor herein has succeeded in discovering a novel human CtBP-binding protein, which is designated CtIP for CtBP-Interacting Protein. The inventor herein has also discovered that CtIP contains the same five amino acid motif (PLDLS) found in adenovirus E1A proteins to which CtBP binds and that deletion of this binding motif in CtIP abolishes its binding to CtBP.
The invention thus provides isolated and purified CtIP polypeptides. A preferred CtIP polypeptide identified herein comprises the human CtIP amino acid sequence shown in FIG. 3 (SEQ ID NO:2).
The present invention also provides isolated polynucleotides encoding a CtIP polypeptide. Preferred polynucleotides identified herein encode the amino acid sequence shown in FIG. 3. A particularly preferred polynucleotide comprises SEQ ID NO:3.
A recombinant cell comprising a polynucleotide encoding for expression a CtIP polypeptide is also within the scope of this invention. The recombinant cell can be used in a method for producing CtIP.
In another embodiment, the invention provides isolated polynucleotides comprising a human nucleotide sequence complementary to a nucleotide sequence encoding a CtIP polypeptide or CtIP fragment. A preferred complementary sequence is SEQ ID NO:4 (FIG. 2B, bottom strand). The invention also provides isolated polynucleotides that specifically hybridize to polynucleotides consisting of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4. These complementary and hybridizing polynucleotides can be used in methods for detecting the CtIP gene and transcription products thereof, as well as in isolating CtIP-encoding polynucleotides from other mammalian and nonmammalian species.
In yet another embodiment, the present invention provides a composition comprising a CtIP polypeptide or fragment and a carrier that facilitates delivery of the CtIP polypeptide or fragment into a target cell.
The present invention also provides polyclonal and monoclonal antibodies that specifically react with CtIP or CtIP fragments and methods for purifying CtIP or detecting its expression using such antibodies.
A method for determining malignancy of a cell in a patient is also provided. The method comprises detecting CtIP expression in the cell, wherein an amount of CtIP expression that is lower than the amount in normal cell indicates the cell is malignant. In one embodiment, the method comprises detecting a CtIP polypeptide with an antibody that specifically reacts with CtIP or a fragment thereof. In other embodiments, the method comprises detecting CtIP mRNA with a polynucleotide probe or by amplifying a target sequence in CtIP mRNA.
In another embodiment, the invention provides a method for inhibiting neoplasia of target cells in a patient which comprises treating the patient with an effective amount of a CtIP polypeptide or fragment. The patient may be treated by administering the CtIP polypeptide or biologically active fragment to the patient or by administering to the patient a polynucleotide encoding the CtIP polypeptide or fragment, through which CtIP or fragment is expressed in the target cells.
The invention also provides a method for identifying agents that inhibit neoplasia of cells which involves determining whether a candidate agent disrupts binding of CtIP and CtBP.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a new protein, CtIP, which is useful in a method for inhibiting neoplasia of cells; the provision of polynucleotides encoding CtIP; the provision of methods for obtaining CtIP by recombinant techniques; the provision of methods for determining the malignancy of a target cell in a patient; the provision of methods for identifying agents that inhibit neoplasia, and the provision of methods that can detect alterations in the CtIP gene.